Degradation of proteins in mammalian cells proceeds via two distinct pathways, the lysosome-dependent and proteasome-dependent systems. The proteasome-dependent system catalyzes the hydrolysis of proteins marked for degradation typically by conjugation to ubiquitin, but also can degrade certain non-ubiquitinated proteins as well.
Proteasome-mediated degradation is also a principal means for controlling the intracellular levels of most cell proteins, including the recognized major regulators of gene expression such as NFκB transcription factor; the inhibitor protein IκBα; hypoxia-inducing factor (HIF)-1a; protooncogenes c-Fos, c-Jun and c-Mos; and the various cyclins. See for example: Whiteside, S. T. and A. Israel, Semin. Cancer Biol. 8: 75-82 (1997); Srinivas et al., J. Biol. Chem. 273: 18019-18022 (1998); Salceda, S. and J. Caro, J. Biol. Chem. 272: 22642-22647 (1997); Huang et al., Proc. Natl. Acad. Sci. USA 95: 7987-7992 (1998); He et al., J. Biol. Chem. 273: 25015-25019 (1998); and Pahl, H. L. and P. A. Baeverle, Curr. Opin. Cell Biol. 8: 340-347 (1996). Furthermore, the smaller-sized peptides generated by the proteasome during the course of protein breakdown are often biologically active; for example, some peptides are presented as antigens on the class I major histocompatibility complex (MHC) [Rock, K. L. and A. L. Goldberg, Annu. Rev. Immunol. 17: 739-779 (1999)]. These degradation products thus can cause different effects and major consequences in a variety of cellular processes, many of which have substantive clinical value.
In particular, NFκB-dependent gene expression is recognized as playing an important role in a number of biological processes of major medical importance including immune, inflammatory and anti-apoptotic responses [Baeuerle, P. A. and D. Baltimore, Cell 87: 13-20 (1996); Beg, A. A. and D. Baltimore, Science 274: 782-784 (1996); and Antwerp et al., Science 274: 787-789 (1996)]. NFκB is a dimer molecule composed of the p50 and p65 (RelA) monomer subunits; and binding of this dimer complex to IκB inhibitor protein in a cytosol is believed to be the main cellular mechanism preventing NFκB-dependent transcription of genes under normal conditions. A number of different extracellular stimuli (including TNFα, I1-1 and lipopolysaccharide) can trigger NFκB transcription factor activation, most notably by causing a rapid degradation of IκB inhibitor protein by the ubiquitin (Ub)-proteasome degradation pathway.
Several steps necessary for proteasome-mediated IκBα degradation to occur have been identified. These include: phosphorylation of IκBα at two sites by a specific IκBα kinase of the SCF1 family [Whiteside, S. T. and A. Ismael, Semin. Cancer Biol. 8: 75-82 (1997); Chen et al., Cell 84: 853-862 (1996)]; the ubiquitination of the phosphorylated IκBα by a specific E3 enzyme complex [Suzuki et al., Biochem. Biophys. Res. Comm. 256: 121-126 (1996); Spencer et al., Genes Dev. 13: 284-294 (1999); Kroll et al., J. Biol. Chem. 274: 7941-7945 (1999); Yaron et al., Nature 396: 590-594 (1998); and Gonen et al., J. Biol. Chem. 274: 14923-14830 (1999)]; and the subsequent binding to VCP (valosin-containing protein) that results in a physical link between the ubiquitinated IκBα and the proteasome.
Separate and distinct from these events is the PR-39 protein. PR-39 is a highly basic arginine/proline-rich peptide originally isolated from porcine intestine on the basis of its anti-bacterial activity [Agerbeth et al., Eur. J. Biochem. 202: 849-854 (1991)]. The PR-39 peptide is secreted in a prepro-protein form that includes a canonical leader sequence and rapidly undergoes cleavage of the N-terminal portion to generate the mature form composed of the 39 C-terminal amino acids [Gudmundsson et al., Proc. Natl. Acad. Sci. USA 92: 7085-7095 (1995)]. While the sequence of the N-terminal part of the prepro-protein is highly homologous to the cathelin gene family members, the sequence of the 39 C-terminal amino acids that make up the mature peptide, has no homology to any other known protein.
Research investigations have shown that PR-39 protein can rapidly cross cell membrances; and, by virtue of its proline-rich composition, may interact with SH3 domains of p47phox and p130Cas [Ross et al., Proc. Natl. Acad. Sci. USA 93: 6014-6018 (1996); and Chan, Y. R. and R. L. Gallo, J. Biol. Chem. 273: 28978-28985 (1998)]. The PR-39 peptide (predominantly produced by blood-derived macrophages) is found at the sites of active inflammation including skin wounds and myocardial infarction and is seen as playing an important role by inducing expression of heparan sulfate-carrying core proteins, syndecan 1 and 4 [Li et al., Circ. Res. 81: 785-796 (1997); and Gallo et al., Proc. Natl. Acad. Sci. USA 91: 11035-11039 (1994)] and inhibiting degradation of the hypoxia-inducible factor (HIF)-1α protein. However, the molecular events and mechanism of action involved in this peptide's actions remain largely unknown.
Accordingly, although there have been many investigations, publications, and developments of these various entities, there remains a general ignorance and failure of understanding by research investigators and clinicians alike regarding useful and effective specific means and methods for suppressing NFκB-dependent gene expression on-demand within living cells, tissues, and organs. Thus, while the value and desirability of selectively controlling NFκB transcription factor activity—especially within cells at localized tissue areas on an as-needed basis for individual subjects—is well recognized, these aims have remained a long-sought goal yet to be achieved to date in a practical manner.